Pooling and Annealing

Pooling and annealing is a common need for NGS applications. One common example is amplicon sequencing using oligonucleotide pools synthesized for targeted genomic regions.  Most often created by combining multiple oligos all at the same concentration, oligo pools can easily contain hundreds of oligos. High specificity assays can require combined oligos to be present at varying concentrations as well. Regardless of the complexity or size of your oligo pool needs, an oligo panel can be prepared utilizing our ISO-certified lab procedures to ensure batch to batch reproducibility.

1. Confidence in results with Individually QC’ed oligos: the oligos that are included in your pool are synthesized in an array format and individually QC’ed. This ensures that there is a representation of each of every oligo of the designed oligos in the pools. Furthermore, our experts can assemble pools of any size at the desired concentration. Since there are no primer dropouts, you can be confident that all the regions are effectively represented in your targeted amplicon sequencing project.
2. Reduction in Costs with lower synthesis scales and multiplexing: since oligonucleotides can be synthesized at lower synthesis scales, through our proprietary technology, than traditional array-based high-throughput synthesizers, it reduces the internal cost which can be passed along to the customer. Additional price savings can be realized by including molecular barcodes in these primers to allow for sample multiplexing.

These pools can be used for ultra-deep sequencing of PCR products for efficient variant identification and characterization. These pools can be designed to interrogate various genes or regions of the genome, including genealogical studies, carrier screening, noninvasive prenatal testing (NIPT), and much more.

Multiplexing

Multiplexing is a simpler version of pooling. It typically involves combining 2 plates together, one plate for the forward primers and the other for the reverse primers, with the well numbers matching up.

• Multiplex Library Prep solutions with Eurofins Genomics Oligonucleotides: Multiple pairs of individually QC’ed, dual-indexed primers with NGS grade service can be annealed and pooled for use in Multiplex Library preparation. This protocol will ensure lower crosstalk between sequence data obtained from different samples.
• Forward and Reverse PCR Primer mixing: Equal molar concentrations of your primers are prepared separately and then mixed.

Our Production team offers a wide variety of customization options, including specific tubes or plates for delivery. Contact us (below) if you’d like more details and a quote. Our Special Projects representative will contact you with additional details.

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Initial NGS strategies generated very large amounts of genomic data that while somewhat useful, did not provide deep coverage to specific genomic regions of interest. To eliminate these data, protocols that allow for genomic targeting, or “capture”, of certain regions of interest were developed. These target enrichment strategies have become extremely useful to those who are examining numerous individuals for differences in specific genomic regions, such as single nucleotide polymorphisms or copy number variation.  There are a number of protocols that are differentiated based upon the way in which the target region is captured.

Related

• Hybridization panel
• Capture Panels
• Assays
• PCR
• Biotin modified EXTREmers

Hybridization

Hybridization-based assay utilize the fragmentation of DNA followed by the ligation of known adapter sequences. The adapters will contain sequence “barcodes” that are unique to each samples which can be searched for and extracted bioinformatically. These ligated sequences are then denatured and subsequently annealed with DNA or RNA single-stranded probes—most of which are biotinylated. Biotinylated allows for the annealed duplex to be captured via streptavidin beads, and after treatment leaves a many fold enrichment of targeted samples compared to the initial starting material. At Eurofins, we can work with you on your Hybridization probe needs. We have the capabilities to synthesize capture panels of probes up to 140 nucleotides in length along with the Biotin modification.

Biotin-labeled EXTREmers

EXTREmers are long oligos up to 200 mer which are synthesized using high-fidelity chemistry on our exclusive new synthesis technology to ensure that the sequence is the most faithful representation of the ordered sequence. Biotin-labeling of these EXTREmers at the 5¢-termini makes these oligonucleotides particularly suited for target enrichment in NGS workflows. Furthermore, we can pool several of these biotin-labeled EXTREmers targeting a region or loci of interest to generate a capture panel. The capture panel can be designed to target genes related to a particular disease, e.g., cancer, inherited diseases, etc., or a specific region, e.g., exome, mitochondria, etc. 

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Continue reading about enrichment panels

Custom UDI Primer Sets

Eurofins Genomics offers custom UDI primers and adaptors to tailor-fit your needs. Our proprietary technology and expertise ensures high accuracy and reproducibility across a variety of projects, from simple indexing to complex multiplexing.

Our process begins with our high-fidelity synthesizers that can produce oligos up to 200bases in length, at both small to intermediate scales. The Hi-Fi synthesis coupled with workflows that spatially and temporally separate oligos significantly minimizes the chances for well-to-well contamination.

Eurofins Genomics is quickly becoming the primer provider of choice for NGS-based commercial assays. Customers have been extremely pleased with the performance of our custom UDIs compared to their existing solution, which has driven organic growth.

The expansion of NGS technology has fueled demand for adaptors and indexing primers on a macro level. UDI primers are a critical component in NGS library preparation. Since its first introduction in the mid-2000s, Next-Generation Sequencing (NGS) has become significantly cheaper and more widely utilized. Uses for NGS range broadly, from whole genome sequencing to targeted DNA/RNA sequencing. The COVID-19 pandemic brought the technology into the mainstream as NGS was utilized for viral genome analysis for variant detection. The increased usage of NGS-based technologies has brought the need for high throughput solutions. Unique Dual Indexes (UDIs) allow for the multiplex sequencing on NGS platforms and subsequent demultiplexing during data analysis. Multiplex sequencing requires highly pure, low cross-contaminated indexes as a single swap may lead to cross-talk.

Universal UDI Primer Sets

Although many companies choose to design their own, custom UDI primer sets and adaptors, other customers are looking for a set of universal, proven UDI Primers to use with Illumina. Due to the increase in demand for custom UDIs, we have reallocated resources from synthesizing stock UDIs onto building custom indexing primers. Stock UDI sets should return to the website towards the end of 2022.”

Eurofins Genomics' NGS UDI Primer Sets to eliminate misassignment by index hopping.

• Ideal for all NGS libraries types including libraries produced with NEB adapters
• Unique pairs of u5 and u7 high performance index primers
• UDIs for standard indexing (8 nucleotides)
• Very high performance also for low-plexity pools
• Minimum edit distance of three bases or higher

Each primer pair has a concentration of 10µM provided liquid in 10µl for single use.

5` linker sequence for target-specific primers:

u5 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'

u7 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'

Note: U5 and U7 contain the complete adapter, whereas I5 and I7 are the short indices of Illumina.

NGS UDI Primer Sets are delivered in a single-use fully-skirted 96-well PCR plate with a pierceable foil seal. Just select your required NGS UDI Primer Set with required quantity and press "Add to Cart". The general structure of the primers can be found in each plate assignment document below.

NGS UDI Primer Sets Out of stock - New versions coming in Q3 NGS UDI Primer Set 96-1 - Click here to download plate assignment with u7 and u5 index sequences and expected u7 and u5 index reads. NGS UDI Primer Set 96-2 - Click here to download plate assignment with u7 and u5 index sequences and expected u7 and u5 index reads NGS UDI Primer Set 48 - Click here to download plate assignment with u7 and u5 index sequences and expected u7 and u5 index reads. NGS UDI Primer Set 24-1 - Click here to download plate assignment with u7 and u5 index sequences and expected u7 and u5 index reads. NGS UDI Primer Set 24-2 - Click here to download plate assignment with u7 and u5 index sequences and expected u7 and u5 index reads.

NGS Adapters and Unique Molecular Indices (UMI)

Unique Molecular Indices (UMIs) are small unique sequences that can be added to adapter oligos to specifically identify particular samples in a given library. UMIs greatly increase the accuracy of quantifying transcripts that are present in libraries at low levels. They can also accurately identify the presence of PCR duplication artifacts. Usually, libraries need to be amplified in order to increase the material that is generated after the genomic DNA is fragmented. The presence of any inherent biases in the amplification of certain genomic regions may incorrectly under or over represent the true abundance in the library. UMIs can be used to tag these genomic regions prior to their amplification, and have their ratios measured after an NGS sequencing run to much more accurately display an estimate of their concentration in the original sample.  UMIs can be used in wide range of applications, such as identifying low-frequency variants, de-duplication of reads, and more.

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